This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As a response to limitations of the descriptive nature of the results from hypothesis-driven, multiple-stage MS (HMS-MS) phosphorylation studies on S. cerevesiae Ace2 and other proteins, we devised a simple, low-cost approach to quantifying changes in phosphorylation using metabolic isotope labeling and mass spectrometry. In this approach, a promoter-replacement strategy is used in S. cerevesiae to genomically integrate the inducible GAL1 promoter upstream of a protein of interest in order for its controllable overexpression. Such an overexpressing strain can be grown in medium containing heavy, stable-isotope labeled amino acids to produce overexpressed, isotope labeled protein that can be used as an internal standard for the quantitative comparison of the phosphorylation states of the protein of interest under multiple different conditions. The overexpression strategy increases the amount of protein obtained per unit isotope label to reduce accordingly the cost of isotope per experiment (which otherwise could be prohibitive). As a test of the method, we investigated the phosphorylation of S. cerevesiae Ace2 (a cell cycle-regulated transcription factor implicated in asymmetric cell division) under different kinase deletion conditions. We had previously discovered phosphorylation of Ace2 at Ser 122, which was potentially a target of the kinase Cbk1. We used the overexpression isotope doping strategy outlined above to quantitatively compare the levels of phospho Ser 122-contatining peptide in Ace2 derived from wild type and cbk1? cells. We found the phosphorylation level to be 2.2 +/- 0.2 times higher in the wild type strain than the cbk1? strain, indicating at least a partial dependence of Ser 122 phosphorylation on Cbk1. We also quantitatively compared multiple sites of phosphorylation of Ace2 (previously implicated as a cyclin-dependent kinase substrate) under conditions where various cyclins were deleted. Initial results indicate that deletion of the cyclins Clb1, Clb2, Clb3 and Clb4 reduce but do not eliminate Ace2 phosphorylation. A manuscript describing this work is in preparation.